The sarcoplasmic reticulum (SR) of striated muscle is comprised of two major compartments, the terminal cisternae, which contain the Ca2+ channels (ryanodine receptors) that open to initiate contraction, and the network SR, which contains much of the Ca2+-ATPase (SERCA) responsible for removing Ca2+ from the myoplasm, leading to relaxation. In mammalian skeletal muscle, these compartments are aligned stereotypically around each sarcomere, with terminal cisternae at the level of the A-I junctions and network SR surrounding M-bands and Z-disks. How these two compartments become organized in this way is poorly understood. Here we focus on the network SR compartment and, in particular, on the possible role in its organization of two proteins, a small (~17 kDa) form of ankyrin, and obscurin, a massive (~800 kDa) protein of the titin superfamily. The small ankyrin, which we refer to as sAnk1 (it is also known as Ank1.5), is encoded by the ANK1 gene and concentrates in the network SR, where it is targeted by its hydrophobic N-terminal sequence thru a mechanism that is not well understood. It is oriented there with its C-terminal region exposed to the myoplasm, where it can bind obscurin. Obscurin is concentrated at the periphery of M-bands and Z-disks, where it is ideally situated to interact with the network compartment of the SR. Consistent with this, the C-terminus of obscurin binds with high affinity to the cytoplasmic domain of sAnk1. We hypothesize that this binding is both necessary and sufficient for the network SR to organize around the sarcomere. We will test this hypothesis in 4 aims: (1) to characterize the binding site on obscurin for sAnk1;(2) to model the 3D structure of obscurin's binding site for sAnk1 in the free and bound states;(3) to determine the basis for the specific targeting of sAnk1 to the network SR;(4) to determine the effect of reducing sAnk1 levels, or altering its ability to interact with obscurin, on the structure and function of the network SR, and its alignment with the contractile apparatus. Our experiments should reveal some of the basic mechanisms responsible for the organization of the SR. Our results should also reveal how changes in cytoskeletal structures associated with internal membranes of striated muscle can lead to myopathies. PUBLIC HEALTH RELEVANCE. For skeletal muscle to function properly, it must organize and stabilize the cellular structures that store and release calcium ions. Defects in these structures have been linked to myopathies and muscular dystrophies, but how they function in muscle is still poorly understood. This proposal is to study the structures in skeletal muscle that store calcium ions.